ABC antibody staining (DAB)

Virtualab Protocols (Chang Bioscience)
The following protocol is for the VECTASTAIN ABC KIT (Vector Lab).

1. Fix and stain cells as described in Staining for immunohistochemistry or FACS , except that the secondary/last antibody is coupled with horseradish peroxide.

2. Make A + B solution: 10 ml A + 10 ml + 980 ml PBS. Mix and let stand at room temperature.

ABC staining is brown. The color changes to blue if the staining buffer contains 1% nickel chloride or dark if 0.5% cobalt chloride.

3. Add A + B solution to the cells, incubate at room temperature for 30 minutes.

4. Wash three times with PBS over a peroid of 30 minutes with gentle rock.

5. Remove PBS, add DAB solution (0.5 mg/ml diaminobenzidine in PBS). Add 30 ml of 0.3% H2O2 per ml of DAB solution. DAB should be protected from light and stock solution stored at -20 C.

6. Observe the extent of staining under a microscope and, when the color is appropriately developed, stop the reaction by washing with several changes of PBS.

DAB is mutagenic, deactivated with bleach.

7. Dehydrate and mount.